Effect of Sperm DNA Integrity on Fertilization Success and Early Embriyonic Development in Unexplained Infertility Cases

Abstract

In almost 15% of infertility cases infertility related pathology cannot defined, and conventional pregnancy is prevented by unexplained factors. Investigations evidenced that sperm DNA damage adversely affects the outcome of assisted reproduction techniques (ART). Recently, non-invasive methods have been developed to select nucleus integrity protectedsperm cells. However; the effects of these methods on unexplained infertility cases remain controversial. In this study the usage of density gradient centrifugation (DG) alone and together with magnetic activating cell sorting (MACS) for sperm separation were compared and the effect of selected spermson embryonic development were investigated. In couples withunexplained infertility after DG and DG+MACS, sperm morphology and DNA quality were analyzed using TUNEL and Hematoxylin Eosin staining methods, respectively. Obtained sperms were followed up by ICSI to observe their potential for fertilization and embryo development. According to our findings, sperm DNA fragmentation and vacuole positivity were significantly lower in MACS + DG treated group as compare to DG treated group. There was no significant difference in fertilization and embryo development potential between oocytes microinjected with sperm washed with DG or MACS+DG which might be due to lack of natural sperm selection in intracytoplasmic sperm injection. Therefore, our data suggest that; MACS may be more effectively used for natural based sperm selection techniques, including; IUI and IVF, instead of ICSI.

Keywords

• Sperm DNA Damage,MACS,DG,TUNEL,Hematoxylin Eosin

Nedeni Açıklanamayan İnfertilite Olgularında Sperm DNA Bütünlüğünün Fertilizasyon Başarısı ve Erken Embriyoner Gelişime Etkisi

Abstract

İnfertilite olgularının yaklaşık %15’inde infertilite sebebi olabilecek bir patoloji saptanmayıp, açıklanamayan sebeplerle konvansiyonel gebelik gerçekleşmemektedir. Araştırmalar, sperm DNA hasarının üremeye yardımcı tekniklerin (ÜYT) sonucunu olumsuz etkilediğini ortaya koymuştur. Çekirdek bütünlüğü korunmuş olan spermleri seçmeye yönelik non-invaziv yöntemler geliştirilmekle birlikte, bu yöntemlerin açıklanamayan infertilite olgularında ÜYT başarısı üzerindeki etkisi henüz tartışmalıdır. Bu çalışmada DNA hasarlı spermlerin dansite gradient santrifüjü (DG) yöntemi ile tek başına ve non-invaziv manyetik aktivasyonla hücre ayırma (MACS) yöntemiyle birlikte eliminasyonu sonrası kullanılan spermlerin fertilizasyon ve erken embriyoner gelişime etkisi karşılaştırılarak uygun semen hazırlama yönteminin saptanması hedeflenmiştir. Açıklanamayan infertilite tanısıyla ÜYT programına alınan çiftlerde, DG yönteminin tek başına ve MACS yöntemiyle birlikte kullanımının, kaliteli sperm elde etmedeki başarısı, her iki yıkama metoduyla elde edilmiş spermlerde TUNEL yöntemi ile DNA bütünlüğü açısından ve Hematoksilen Eosin boyamasıyla morfolojik açıdan değerlendirildi. Klinik parametrelere etkisini değerlendirmek amacıyla, her iki yıkama yöntemiyle elde edilen spermlerin ICSI sonrası fertilizasyon ve embriyo gelişimsel potansiyeline bakıldı. MACS+DG yöntemiyle yıkanan sperm örneklerinde sperm DNA fragmantasyon oranının ve vakuol (+) sperm oranının DG yöntemiyle elde edilen spermlere kıyasla istatistiksel olarak anlamlı düzeyde azaldığı görüldü. DG veya MACS+DG ile yıkanan spermlerle mikroenjeksiyonu gerçekleştirilen oositler arasında fertilizasyon ve embriyo gelişimsel potansiyelinde bir farklılık oluşmaması intrasitoplazmik sperm enjeksiyonu uygulamalarında spermin doğal seçiliminin söz konusu olmamasının bir sonucu olduğu düşünülerek, MACS tekniğinin ICSI protokollerinde gerekli olmadığı, spermin doğal seçiliminin gerçekleştiği intrauterin inseminasyon (IUI) ve IVF uygulamalarında daha etkin olacağı sonucuna varıldı.

Keywords

• Sperm DNA Hasarı,MACS,TUNEL,, HEMATOKSİLEN EOZİN,DG

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